The DNA amplification technique represented by polymerase chain reactions (PCR); Nature, vol. 324 (6093), 1986, pp.13-19) is a well-known technique in the field of molecular biology. Detection, analysis, transcription or amplification of nucleic acids by the PCR technique is one of the most important operations in modern molecular biology and is especially important in gene expression studies, diagnosis of infectious agents or hereditary diseases, cDNA production, analysis of retroviruses, etc.
Since DNA amplification performance depends on the performance of the DNA polymerase used, various DNA polymerases have been searched for in nature or improved. For example, PCR was originally performed using insufficiently thermostable DNA polymerases derived from mesophiles such as E. coli. However, since PCR is performed under highly stringent conditions, i.e., themocycling at temperatures in the range of about 23° C. to about 100° C. many times, the success rate (probability with which object DNA is successfully amplified) was low. It is now, however, common practice to utilize highly thermostable thermophile-derived DNA polymerases.
One of the important performances required of DNA polymerases is “elongation rate”. A typical method of determining DNA elongation rate comprises performing a DNA synthesis reaction in a buffer using DNA prepared by annealing of M13 single-stranded DNA (1.6 μg) and complementary primer(s) (16 pmole) as a template and using KOD, Pfu, Deep Vent, Taq or like various DNA polymerases (5U) (as an enzyme), followed by calculating the DNA elongation rate from the relationship between the reaction time and the size of DNA synthesized.
For example, the DNA elongation rate of KOD polymerase is 105 to 130 bases/second, that of Pfu polymerase is 24.8 bases/second, that of Deep Vent polymerase is 23.3 bases/second, and that of Taq polymerase is 61.0 bases/second (Takagi, M. et al.: Characterization of DNA polymerase from Pyrococcus sp. KOD1 and its application to PCR; Appl. Environ. Microbiol. 63, 4504-45410, (1997)).
“Fidelity” is another important performance required of DNA polymerases. One example of a method for evaluating the DNA synthesis fidelity of DNA polymerase is using a ribosomal protein S12 (rpsL) gene associated with streptomycin resistance. Streptomycin is an antibiotic that inhibits protein synthesis in prokaryote. This antibiotic binds to bacterial 30S ribosomal RNA (rRNA) to inhibit initiation complex for protein synthesis formation reactions and cause the misreading of genetic code. Streptomycin-resistant strains have a mutation at the ribosomal protein S12 locus. This mutation is known to produce pleiotropic effects such as inhibiting suppressor tRNA from reading the stop codon to enhance translation fidelity of the ribosome. Thus, when PCR amplification is carried out using rpsL gene as a template, a mutation is introduced with a certain probability. When the mutation occurs at the amino acid level, the rpsL protein structure is changed so that streptomycin cannot act on 30S ribosomal RNA (rRNA). Therefore, when the amplified plasmid DNA is used to transform E. coli, the more mutations introduced, the higher the frequency of streptomycin-resistant strain appearance.
The plasmid pMol 21 (described in Journal of Molecular Biology (1999) 289, 835-850) is a plasmid containing rpsL gene and ampicillin resistant gene. A primer set for PCR amplification (one of the primers is biotinylated and has a MluI restriction site introduced therein) is designed on the ampicillin resistant gene of the plasmid pMol 21 to amplify the full-length plasmid by PCR using a thermostable DNA polymerase. The obtained PCR product is purified using streptavidin beads and cut out using the restriction enzyme MluI, followed by ligating the ends using DNA ligase to transform E. coli. The transformant is inoculated into two types of plate media, i.e., one containing ampicillin, and the other containing both ampicillin and streptomycin. The ratio of numbers of colonies appearing on the two plate media is calculated to determine the fidelity or correctness of DNA synthesis (Kunkel, Journal of Biological Chemistry, vol. 260, 1985, pp.5787-5796).
When PCR product fidelity of Taq DNA polymerase is determined by the above method of determining DNA synthesis fidelity, the mutation rate was 4% or more. In the case of a DNA polymerase capable of exhibiting 3′-5′ exonuclease activity when used alone, the mutation rate was 0.05 to 1%. When using a mixed enzyme of an enzyme not having 3′-5′ exonuclease activity such as Taq DNA polymerase and an enzyme having 3′-5′ exonuclease activity, the mutation rate was 2 to 4%. The mutation rate achieved by KOD DNA polymerase was 0.1% or less. This DNA polymerase is the most suitable enzyme for obtaining high-fidelity PCR products.
To what length the target DNA can be amplified (hereinafter referred to as “long-PCR performance”) is also an important requirement of DNA polymerases.
DNA polymerases, kinds of thermostable (or heat-resistant) DNA polymerases, are roughly classified into Pol I-type enzymes represented by Taq DNA polymerase and Tth DNA polymerase, and α-type enzymes represented by Pfu DNA polymerase. Generally, Pol I-type enzymes achieve high DNA elongation rates but have poor fidelity because of the lack of 3′-5′ exonuclease activity. In contrast, α-type enzymes, which possess 3′-5′ exonuclease activity, have high fidelity but achieve low DNA elongation rates. Thus, although the two types of DNA polymerases have excellent properties in “elongation rate” and “fidelity”, respectively, neither have both.
With the purpose of combining the merits of both types of enzymes, Proc. Natl. Acad. Sci. USA, vol.91, 1994, pp.2216-2220 describes a technique utilizing a mixed type enzyme prepared by mixing two kinds of thermostable enzymes. This enzyme mostly comprises a Pol I-type enzyme, which is considered to mainly perform DNA biosynthesis, while the α-type enzyme merely proofreads base errors.
Japanese Patent Publication No. 3112148 describes KOD DNA polymerase, a single enzyme (α-type enzyme) having both excellent “elongation rate” and “fidelity”.
A method using HindIII-digested λDNA labeled with 3H at the 3′ end as a substrate and measuring the rate of 3H release under the optimal temperatures for each polymerase is known as a typical method of evaluating 3′-5′ exonuclease activity. More specifically, using a HindIII-digested λDNA fragment having [3H] TTP incorporated therein as a substrate, for example, the DNA fragment and a polymerase are left in a buffer solution (20 mM Tris-HCl pH 6.5, 10 mM KCl, 6 mM (NH4)2SO4, 2 mM MgCl2, 0.1% Triton X-100, 10 μg/ml BSA) under the optimal conditions for the polymerase and the [3H]TTP release rate is determined. The substrate DNA is prepared by adding 0.2 mM of dATP, dGTP, dCTP and [3H] TTP to 10 μg of HindIII-cleaved λDNA and extending the 3′ end with Klenow polymerase, followed by phenol extraction and ethanol precipitation to recover the DNA fragment and further purification by removal of the released mononucleotides using a spun column (product of Clontech).
To improve DNA amplification performance, various studies have been conducted to improve the composition of DNA synthesis reaction solutions. Examples of tested buffers include Tris, Tricine, bis-Tricine, HEPES, MOPS, TES, TAPS, PIPES, CAPS, etc. Examples of tested salts include potassium chloride, potassium acetate, potassium sulfate, ammonium sulfate, ammonium chloride, ammonium acetate, magnesium chloride, magnesium acetate, magnesium sulfate, manganese chloride, manganese acetate, manganese sulfate, sodium chloride, sodium acetate, lithium chloride, lithium acetate, etc. Examples of tested additives include DMSO, glycerol, formamide, betaine, tetramethylammonium chloride, PEG, Tween 20, NP40, ectoine, polyols, E. coli SSB protein, phage T4 gene 32 protein and BSA, etc (Published Japanese Translation of PCT International Publication of Patent Application No. H9(1997)-511133, U.S. Pat. No. 5,545,539, International Publication No. WO 96/12041, U.S. Pat. No. 6,114,150, WIPO Publication No. WO 99/46400, Nucleic Acids Research, vol. 23, 1995, pp.3343-3344, and Nucleic Acids Research, vol. 28, 2000, p.70).
However, the above reagents are not effective for all DNA polymerases. Effectivity depends on the enzyme. Therefore, different reaction solution compositions have been investigated for different enzymes, and optimal reaction buffer solutions for individual enzymes have been recommended. It has been considered impossible to achieve a higher amplification efficiency than commercially co-packaged buffer solutions.
With the recent advances in biotechnology, the required performance levels in DNA amplification, especially PCR, of “elongation rate”, “fidelity” and “long-PCR performance” have been raised ever upwards. In particular, DNA amplification fidelity has become more important in that successful elucidation of the entire human genome sequence has shifted the focus of study from mere detection of genes or analysis of their differences to analysis of the functions of genes or proteins encoded by the genes.
Therefore, the establishment of a DNA amplification method achieving these requirements has been desired, but none of the prior art is satisfactory for practical use in all the requirements “elongation rate”, “fidelity” and “long-PCR performance”.